Methods in Molecular Biology • 16 Enzymes of Molecular Biology

(Nancy Kaufman) #1

Ligases 215


The enzyme will not ligate a 3' phosphate group to a 5' hydroxyl, a
3' hydroxyl to a 5' hydroxyl, a 3' dideoxy nucleoside to a 5' phosphate,
or a 3' hydroxyl to a 5' triphosphate terminus. If only one of the 5'
termini of a double-stranded break is phosphorylated, DNA ligase can
only rejoin that one strand and the resultant product is a nicked mol-
ecule, since the second strand cannot be ligated. However, this double-
stranded DNA molecule is intact and can then be used in transformation
reactions enabling the ligation to be completed in vivo.
Only T4 DNA ligase will ligate blunt-ended DNA molecules, the
reaction proceeding by way of nicked intermediates (14). Unlike join-
ing cohesive ends, the rate is not linearly dependent on enzyme con-
centration. The enzyme can also ligate at a mispaired 3' base (18,19)
and will ligate DNA/RNA hybrids, but with much reduced activity.
The reverse reaction is also catalyzed (20), the enzyme behaving as
an AMP-dependent endonuclease, yielding nicked DNA. The two
activities of DNA ligase result in the slow (but eventually complete)
relaxation of supercoiled DNA.
The only action on RNA performed by E. coli DNA ligase is the
ligation of the 3' hydroxyl of an RNA strand to a phosphorylated 5'
DNA terminus (21). T4 DNA ligase has some activity in joining RNA
molecules annealed to DNA and even RNA:RNA (22).
E. coli DNA ligase has a K,,, for the 5' phosphate group of 2.5-5.6
× 10-8M (23). The value for T4 DNA ligase is 6 × 10-7M for cohesive ends
(24), 5 × 10-SM for blunt ends, and 1.5 × 10-9M for repairing nicks (17).


2.2.2. Cofactors
DNA ligase requires a nucleotide cofactor for reaction, forming a
covalent AMP-enzyme intermediate. E. coli DNA ligase utilizes NAD
(as do ligases from B. subtilis, T. thermophilus, and S. typhimurium).
The enzyme has a high specificity for NAD with a Km of 3 x 10 -8 to
7 × 10-6M (25).
T4 DNA ligase (and T7 and mammalian ligases) requires ATP. It
will also utilize dATP (at 0.5% of the rate), which acts as a competitive
inhibitor with ATP (17). The value of K,n ATP for the joining reaction
is 1.4-10.0 × 10-SM and the K i dATP is 3.5 × 10-SM. For the pyrophos-
phate exchange reaction used in the enzyme assay (17), K m ATP is 2 ×
10-6M and K i dATP is 1 × 10-SM (1). T7 DNA ligase can in fact utilize
either ATP or dATP (26).

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